Sorption reinforced catalytic coating system and method for the degradation of threat agents

ABSTRACT

Sorption reinforced catalytic coating system for the degradation of threat agents including a synzyme coating about a material, the synzyme coating having bucket-shaped molecules for the sorption and degradation of the threat agents. A binding agent is configured for synzyme immobilization to maximize loading and retention of the synzyme coating on the material.

RELATED APPLICATIONS

This application hereby claims the benefit of and priority to U.S.Provisional Application Ser. No. 61/281,900, filed on Nov. 24, 2009under 35 U.S.C. §§119, 120, 363, 365, and 37 C.F.R. §1.55 and §1.78,incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to a sorption reinforced catalytic coating systemand method for the degradation of threat agents.

BACKGROUND OF THE INVENTION

Chemical, Radiological, Biological and Nuclear Defense (CRBN) is a highpriority of the Army and National Security due to rogue states and therise of terrorist organizations that are technologically sophisticated,well-financed and committed to inflicting damage on U.S. interests andpersonnel. The increased threat of chemical and biological agent attackin various military theaters combined with the possibility of terroristattacks on the general public has led the inventors hereof to evaluateprotective equipment for military and civil defense. Battlefieldchemical protective materials are sought that do not just serve assimple barriers to incoming threats, but can reactively decontaminatethe threat agents so as not to impede the mission at hand.

It is known that an extremely small amount of a CWA, such as a nerveagent, can affect the transmission of chemical nerve impulses in humansleading to death soon after exposure. Because of this toxicityprotection against CWAs, biological weapons and toxic industrialchemicals (TICs) requires capture efficiency greater than about 99.97percent combined with sufficient decontaminating capabilities

There are several known decontaminating technologies that have beendeveloped to degrade CWAs and TICs. However, to date, there is no singlesolution to provide comprehensive protection for personnel working inboth dry and wet environments.

One known chemical method typically employed to degrade stockpiles ofCWAs uses caustic salts at high temperatures. However, such a methodoften generates side-reactions which can reverse to form the originaltoxic chemical agents. To overcome these shortcomings inorganiccatalysts have been used to degrade CWAs and TICs because they are morerobust but have low catalytic activity. More recently biologicalcatalysts have been developed which rapidly and safely decontaminateCWAs or TICs but have limited life spans because of their fragilenature.

Enzymes, such as organophosphate degrading enzymes (OPH, OPAA), and thelike, are reported to be efficient catalytic materials that effectivelydegrade CWAs and TICs. These enzymes denature rapidly when in solutionand their performance is based solely on limited temperature and pHranges. To mitigate these shortcomings the enzymes need to beimmobilized, which will allow the system to be reusable, recyclable andrecoverable while maintaining chemical activity towards all types ofCWAs and TICs in both dry and wet environments.

Several known methods for immobilization of enzymes often rely onstabilizing the enzymes during the deposition phase and retard theirdeactivation upon prolonged exposure to stressful conditions. One knownmethod relies on a covalent chemistry approach in which the reactionconditions use organic solvents. However, such a technique causes amajor loss of enzyme activity. In one example, a research groupdeveloped a polyurethane nanosponge to degrade toxins. However, most ofthe enzymes incorporated into the sponge were eventually renderedinactive during the polymerization process.

To avoid such a loss of activity of the enzymes, one knownlayer-by-layer (LBL) method provides a versatile platform for thefabrication of multifunctional bio-materials utilizing catalyticenzymes. A Naval Research Laboratory (NRL) research team has reportedenzymes immobilized in polyelectrolyte multilayer assemblies capable ofmaintaining their catalytic activity over long periods of time, e.g.,greater than about 8 months. Fabricated OPH enzyme-bearing cotton clothsvia layer-by-layer assembly preserved their hydrolytic activity againstmethyl parathion (MPT) within 5 minutes of exposure. The non-covalentmethod of the NRL team for incorporating enzymes helps tremendously inmaintaining enzyme activity by protecting the enzyme from denaturizing.However, even this layer-by-layer approach has some distinct drawback.Many catalytic enzyme multilayers were shown to have limited activitytowards their respective agents. This indicates only a limited amount ofthe enzyme, due to its amphoteric nature, was actually loaded onto thesystem.

BRIEF SUMMARY OF THE INVENTION

This invention features a reinforced catalytic coating system for thedegradation of threat agents including a polyurethane coating about amaterial configured to provide loading and stabilization of one or moreenzymes and for the sorption of the threat agents. An enzyme coatingincluding the one or more enzymes is disposed about the polyurethanecoating and is configured to degrade the threat agents. A binding agentis configured for enzyme immobilization to maximize loading andretention of the one or more enzymes on the enzyme coating.

In one embodiment, the polyurethane coating may be functionalized withorganic bucket-shaped molecules configured to stabilize the one or moreenzymes of the enzyme coating. The bucket-shaped molecules may includecyclodextrin. The cyclodextrin and derivatives thereof may includeβ-cylclodextrin. The polyurethane coating may be functionalized withchemical groups configured to stabilize the one or more enzymes of theenzyme coating. The chemical groups may include sugar groups. The sugargroups may include trehalose. The polyurethane coating may befunctionalized with calixarene and derivates thereof configured for thesorption of radiological threat agents. The polyurethane coating may befunctionalized with chemical groups to promote water scavenging. Thechemical groups may include trehalose. The enzyme coating may includeorganophosphate degrading enzymes. The organophosphate degrading enzymesmay include one or more of organophosphorous hydrolase (OPH),organophosphorous acid anhydrolase (OPAA) and haloalkane dehalogenase(HD). The vaporized binding agent may include vaporous glutaraldehyde.The glutaraldehyde may be vaporized. The glutaraldehyde may beconfigured to selectively attach to the enzyme coating. Theglutaraldehyde may be configured to attach to the enzyme coating toprevent delamination. The binding agent may be configured to provide forrepeated cleaning cycle of the coating system. The binding agent may beconfigured to provide for reusability of the coating system. The enzymecoating may be configured to degrade the threat agents in humidenvironments. The material may includes one or more of fiber basedfabrics, meltblown nano based fabrics, electrospun nano fibers, cotton,and/or nylon. The coating system about the agent may be configured tomake protective clothing. The enzyme coating may be exposed to achaperone configured to enhance refolding of the one or more enzymes.

This invention also features a method for making a reinforced catalyticcoating system for the degradation of threat agents, the method includescoating a material with a polyurethane coating configured to provideloading and stabilization of one or more enzymes and for the sorption ofthe threat agents. The polyurethane coating is coated with an enzymecoating including the one or more enzymes configured to degrade thethreat agent. The material with the polyurethane coating and the enzymecoating is exposed to a binding agent configured for enzymeimmobilization to maximize loading retention of the one or more enzymeson the enzyme coating.

In one embodiment, the method may include the step of functionalizingthe polyurethane coating with organic bucket-shaped molecules configuredto stabilize the one or more enzymes of the enzyme coating. The methodmay include the step of functionalizing the polyurethane coating withchemical groups configured to stabilize the one or more enzymes of theenzyme coating. The method may include the step of functionalizing thepolyurethane coating with calixarene and derivates thereof configuredfor the sorption of radiological threat agents. The method may includethe step of functionalizing the polyurethane coating with chemicalgroups that promote water scavenging. The method may include the step ofproviding the enzyme coating with organophosphate degrading enzymes. Themethod may include the step of vaporizing the binding agent. The methodmay include the step of making protective clothing with the coatingsystem.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Other objects, features and advantages will occur to those skilled inthe art from the following description of a preferred embodiment and theaccompanying drawings, in which:

FIG. 1 is a three-dimensional view showing the primary components of oneembodiment of the sorption reinforced catalytic coating system for thedegradation of threat agents of this invention;

FIG. 2 is a depiction of the structure of β-cyclodextrin used in oneembodiment of the system shown in FIG. 1;

FIG. 3 shows one example of the structure of the binding agent shown inFIG. 1;

FIG. 4 is a three-dimensional view showing the primary components ofanother embodiment of the sorption reinforced catalytic coating systemfor the degradation of threat agents of this invention;

FIG. 5 is a depiction of the structure of trehalose which may be used inone embodiment of the system shown in FIG. 4;

FIG. 6 is a depiction of the structure of calixarene which may be usedin one embodiment of the system shown in FIG. 4;

FIG. 7 is a three-dimensional view showing the primary components of yetanother embodiment of the sorption reinforced catalytic coating systemfor the degradation of threat agents of this invention;

FIG. 8 is a schematic side view showing in further detail the variouscoatings and binding agents shown in FIG. 7;

FIG. 9 is a photograph showing one example of the synzyme coating of oneembodiment of this invention coated on a fabric and being exposed to acolorless methylparaoxon (MPO) toxin and showing the development of adistinctive yellow color of the p-Nitrophenol (pNP degradationby-products);

FIG. 10 is a schematic side view of one embodiment of sorptionreinforced catalytic coating system of this invention showing in furtherdetail one example of an enzyme coating degrading a MPT toxin to producedegraded non-lethal by-products;

FIG. 11 shows one example of the preparation ofpolyurethane-β-Cyclodextrin (Poly-β-CD) using exhaustive cross-linkingof β-CD;

FIG. 12 is a graph showing one example of the absorption behavior of acotton thread coated with a Poly-β-CD in accordance with one or moreembodiments of this invention;

FIG. 13 shows graphs depicting an enzyme coating with an OPH coatedPU-CX particles degrading MPT to pNP in accordance with one or moreembodiments of this invention;

FIG. 14 depicts graphs showing the enhanced catalytic activity of theenzymes of the enzyme coating configured as an OPH coated cotton threadas a result of the refolding process in accordance with one or moreembodiments of this invention;

FIG. 15 depicts one example of the hypothetical catalytic process ofβ-CD-BPEI for the decontamination of the sarin (GB) toxin;

FIG. 16 are photographs depicting electrospun nanofiber mats before andafter lyophilization in accordance with one embodiment of thisinvention;

FIG. 17 depicts the binding of two BPEI molecules using a GA as abinding agent;

FIG. 18 depicts graphs showing one example of the decontaminationkinetics of one embodiment of the sorption reinforced catalytic coatingsystem for the degradation of threat agents of this invention employingan OPH enzyme-polymeric substrates at different time intervals;

FIG. 19 is a photograph showing the enzyme coating of one or moreembodiments of this invention coated on a fabric and being exposed tocolorless toxin and showing the development of a distinctive color ofthe pNP degradation by-products;

FIG. 20 is a graph showing one example of the enzymatic hydrolysis of aSoman toxin exposed to one or more embodiments of the system and methodof this invention employed on a meltblown fabric;

FIG. 21 depicts graphs showing the sorption and decontaminating capacityof OPH coated Poly-β-CD MB fibers in accordance with one or moreembodiments of this invention; and

FIG. 22 shows graphs depicting testing of one embodiment of the sorptionreinforced catalytic coating system for the degradation of threat agentsof one embodiment of this invention which was tested by a third party.

DETAILED DESCRIPTION OF THE INVENTION

Aside from the preferred embodiment or embodiments disclosed below, thisinvention is capable of other embodiments and of being practiced orbeing carried out in various ways. Thus, it is to be understood that theinvention is not limited in its application to the details ofconstruction and the arrangements of components set forth in thefollowing description or illustrated in the drawings. If only oneembodiment is described herein, the claims hereof are not to be limitedto that embodiment. Moreover, the claims hereof are not to be readrestrictively unless there is clear and convincing evidence manifestinga certain exclusion, restriction, or disclaimer.

There is shown in FIG. 1 one embodiment of sorption reinforced catalyticcoating system 10 for the degradation of threat agents of thisinvention. System 10 includes synzyme coating 12 about material 14(discussed below). Synzyme coating 12, shown in further detail in anexploded blowout 13, preferably includes bucket-shaped molecules 16which provide for sorption of threat agents 18, e.g., CWAs, TICs, orsimilar type threat agents. In one example, bucket-shaped molecules 16may include cyclodextrin derivatives thereof, e.g., β-cyclodextrin asshown in FIG. 2. Bucket-shaped molecules 16 may also includeα-cyclodextrin, or any other similar type bucket-shaped molecules knownto those skilled in the art. Bucket-shaped molecule 16 preferablyincreases the residence time of threat agents 18 in synzyme coating 12which increases the efficiency of system 10 to degrade threat agents 18.

In one embodiment, synzyme coating 12 includes a polyimine which ispreferably branched, e.g., branched polyethyleneimine (BPEI) or similartype of polyimine. For example, synzyme coating 12 may include BPEIhaving the chemical structure depicted by BPEI molecule 22 withinbrackets 24 and BPEI molecule 26 within brackets 28. BPEI is preferablyfunctionalized with cyclodextrin and derivatives thereof, e.g.,β-cyclodextrin, FIG. 2, α-cyclodextrin, or any other similar typebucket-shaped molecules known to those skilled in the art. The BPEI insynzyme coating 12, FIG. 1, preferably includes amines, e.g., primaryamine 30 of BPEI molecule 22 which degrade threat agents 18.

In one example, bucket-shaped molecules 16 preferably include an innerhydrophobic pocket, e.g., inner hydrophobic pocket 30, FIG. 2, ofβ-cyclodextrin which attracts and increases the residence time of threatagents 18, FIG. 1, in synzyme coating 12 and expels hydrophilicdegradation products 19 of the threat agents 18.

CWAs and TICs are typically hydrophopic. System 10 may overcome thisproblem by the introduction of the bucket-shaped molecules having ahydrophobic pocket, such as β-cyclodextrin and derivatives thereof whichhelps dissolution and extends the residence time of the CWAs and TICsvia a host-guest complex formation. Through this interaction a morefavorable homogenous environment is formed which allows for improveddegradation of threat agents 18.

System 10 also includes a binding agent configured for synzymeimmobilization to maximize loading and retention of synzyme coating 12on material 14. Synzyme coating 12 on material 14 is preferably exposedto a vaporized binding agent then dried. In one example, the bindingagent may be an aldehyde functionalized binder, such as glutaraldehyde(GA) 34, FIG. 3, and derivatives thereof, where R may be CHO (formyl),CH₂OH (hydroxymethyl), CH₂F (fluoromethyl), CH₃ (methyl), or CH₂SH(mercaptomethyl). The binding agent, e.g., GA-34, selectively attachesto synzyme coating 12 to maximize the loading and retention of synzymecoating 12 to material 14. In one example, GA-34 (with the R as CHO),shown in its reacted state at 35 in box 23, selectively attaches to freeamines of synzyme coating 12, e.g., secondary amines or primary aminesof BPEI molecules 22, 26, respectively. The binding of BPEI molecules22, 26 using GA-34 results in the structure indicated in dashed box 23.

Thus, the binding agent of system 10 maximizes loading and retention ofsynzyme coating 12 on material 14. This allows system 10, e.g., whenutilized to make fiber-based fabrics such as those used in protectiveclothing or similar type articles, to withstand repeated cleaningcycles. The binding agent also prevents delamination of synzyme coating12 which makes system 10 and the fabrics, protective clothing, articles,and the like, manufactured therefrom insoluble to water or similar typefluids. Thus, the protective clothing made using system 10 is reusableand recyclable. System 10 also effectively degrades threat agent 18 in alow moisture environment. Material 14 used by system 10 may includemeltblown nano-based fabrics, electrospun nano fibers, cotton, nylon,and similar type materials.

Sorption reinforced catalytic coating system 40, FIG. 4, where likeparts have been given like numbers, for the degradation of threat agentsof another embodiment of this invention includes polyurethane coating 42about material 44. Material 44 is preferably the same as material 14discussed above. Polyurethane coating 42 provides for the attachment ofone or more enzymes, e.g., enzyme 48 shown in block 47, and for thesorption of threat agents 18.

System 40 also includes enzyme coating 46 about polyurethane coating 42.Enzyme coating 46 includes one or more enzymes which degrade threatagents 18. In one example, enzyme coating 46 may include organophosphatedegrading enzymes, such as organophosphorhous hydrolase (OPH),organophosphorous acid anhydrolase (OPAA), and haloalkane dehalogenase(HD), or similar type organophosphate degrading enzymes. In the exampleshown in FIG. 4, enzyme 48 is a single OPH enzyme. Enzyme coating 46 mayalso include any type of enzyme known to those skilled in the art whichcan be used to degrade threat agents.

System 40 also includes a binding agent configured for enzymeimmobilization to maximize loading and retention of the enzymes inenzyme coating 46 on material 44. Enzyme coating 46 on material 44 ispreferably exposed to a vaporized binding agent then dried. Similar asdiscussed above with reference to FIGS. 1 and 3, the binding agent maybe GA-34. In this embodiment, GA-34 (with R as CHO), shown in itsreacted state at 50 in block 49, FIG. 4, selectively attaches to theenzymes of enzyme coating 46. In this example, the reacted GA-50 bindsenzyme 48 (e.g., a single OPH enzyme) with enzyme 49 (e.g., anothersingle OPH enzyme) to maximize the loading and retention of enzymes 48,49 to enzyme coating 46 on material 44. The binding of the enzymes toeach other on enzyme coating 46 using the binding agent may preventsystem 40 from delaminating and makes system 40 insoluble in water. Theresult is system 40, e.g., when used to make fiber-based fabrics such asthose used in, protective clothing, articles, and the like, as discussedabove, may withstand repeated washings, may be recyclable, reusable andmay effectively degrade threat agents 18 in a wet environment.

In one design, polyurethane coating 42, shown in greater detail in block43, may be comprised of polyurethane backbone 56 which is functionalizedwith organic bucket-shaped molecules discussed above which stabilize theenzymes of enzyme coating 46. In one embodiment, polyurethane coating 42may be functionalized with cyclodextrin and derivates thereof, e.g.,β-cyclodextrin, FIG. 2, shown in this example functionalized topolyurethane coating backbone 56, FIG. 4, at 60. The bucket-shapedmolecules may also include α-cyclodextrin or similar type bucket-shapedmolecules which can be grafted onto the polyurethane coating 42 withsorptive capabilities. In this example, β-cyclodextrin stabilizes theenzymes of enzyme coating 46 by preventing the enzymes fromconformational changes by secondary interactions, such as hydrogenbonding and hydrophobic interaction. β-cyclodextrin and the derivativesthereof may also effectively sorb threat agents 18.

Polyurethane coating 42 may also be functionalized with chemical groupsthat stabilize the enzymes of enzyme coating 46. For example,polyurethane coating 42 may be functionalized with sugar groups, such astrehalose 62, FIG. 5, shown functionalized to polyurethane coatingbackbone 56, FIG. 4 at 64 in block 43. Block 47 shows one example ofenzyme coating 46 with a plurality of plurality of trehalose molecules62 about enzyme 48 which stabilize enzyme 48. The functionalizedpolyurethane coating 42 with sugar group similarly prevents the enzymesin enzyme coating 46 from denaturing.

Polyurethane coating 42 may also be functionalized with calixarene 66,FIG. 6, and derivates thereof, which sorb radiological threat agents 18.Polyurethane coating 42, FIG. 4, may also be functionalized withchemical groups which promote water scavenging, e.g., trehalose 62, FIG.5.

Similar, as discussed above with reference to FIG. 1, system 40 withmaterial 44 may include fiber-based fabrics that may be used inprotective clothing, various articles, and the like. In other examples,material 44 may include meltblown nano-based fabrics, electrospun nanofibers, cotton, nylon, and similar type materials.

Reinforced catalytic coating system 80, FIG. 7, where like parts havebeen given like numbers, is a combination of system 10, FIG. 1, andsystem 40, FIG. 4. In one embodiment, system 80 includes polyurethanecoating 42 about material 44. Similar as discussed above with referenceto FIG. 4, polyurethane coating 42 provides for the attachment of one ormore enzymes and for the sorption of threat agents 18. Polyurethanecoating 42 is preferably functionalized with organic bucket-shapedmolecules discussed above which stabilize the enzymes of enzyme coating46. System 40 also includes enzyme coating 46 about polyurethane coating42 which, similar as discussed above, includes one or more enzymes whichdegrade threat agents 18.

System 80 also includes a first binding agent configured for enzymeimmobilization to maximize loading and retention of the enzymes inenzyme coating 46 on polyurethane coating 42. Enzyme coating 46 onmaterial 44 is preferably exposed to the first binding agent then dried.Similar as discussed above with reference to FIGS. 3 and 4, the firstbinding agent may be GA-34 which, in this example, is shown in itsreacted state at 50, block 49, FIG. 7. The first binding agentselectively attaches to the enzymes of enzyme coating 46 and binds themtogether to maximize loading and retention of the enzymes in enzymecoating 46 on polyurethane coating 42.

System 80 includes synzyme coating 12′, shown in further detail in block51, having a similar structure as synzyme coating 12, FIG. 1. However,in this embodiment, synzyme coating 12′, FIG. 7, is disposed over enzymecoating 46 with the first binding agent. Synzyme coating 12′ preferablyincludes bucket-shaped molecules which provide for sorption anddegradation of threat agents, e.g., threat agents 18 shown in block 51,such as, CWAs, TICs, or similar type threat agents, as discussed abovewith reference to FIG. 1.

System 80, FIG. 7, also includes a second binding agent, e.g., GA-34,FIG. 3, having the structure in its reacted state shown at 53 in box 55,FIG. 7. Synzyme coating 12 on material 14 is preferably exposed assecond binding agent then dried. The second binding agent is preferablyconfigured for synzyme immobilization to maximize loading and retentionof synzyme coating 12′ on enzyme coating 46. In this example, the GA-53has bound synzyme A-61 to synzyme B-63.

FIG. 8 shows one example of one embodiment of the various coatings andbinding agent of system 80: polyurethane coating 42 functionalized, inthis example, with β-cyclodetrin on material 44, enzyme coating 46 withfirst binding agent, indicated at 90, binding enzymes 92, and synzymecoating 12′ with the second binding agent, indicated at 94, bindingsynzyme molecules 96 together. Hydrogen bonding and/or hydrophopicinteractions binds enzyme coating 46 to polyurethane coating 42,indicated at 98. The second binding agent also binds synzyme coating 12′to enzyme coating 46, indicated at 100.

The result is system 80 with polyurethane coating 42 on material 44,enzyme coating 46, the first binding agent which maximizes the loadingand retention of the enzymes of the enzyme coating, synzyme coating 12′and the second binding agent which maximizes retention of synzymecoating 12′ on enzyme coating 46 results in a system which includes allthe features of the two embodiments discussed above with reference toFIGS. 1-6 into a comprehensive system which effectively degrades threatagents in both wet and dry environments. Thus, system 80 can be used tomake fiber-based fabrics for protective clothing, various articles, andthe like, that are insoluble to water, can withstand repeated washing,are recyclable and reusable, and do not delaminate.

EXAMPLES

The following examples are meant to illustrate and not limit the variousembodiments of the present invention.

Novel sorption reinforced catalytic systems, capable of decontaminatingCWAs and TICs, have been conceived and demonstrated.

These new catalytic systems and methods discussed herein may be based onenzyme bearing polymeric composites. These new systems have the addedbenefit of maximizing the enzyme loading while stabilizing the naturallyfragile enzyme to maintain its catalytic activity by preventing itsdenaturing. Unique methods and compositional approaches that producecatalytic systems that chemically degrade CWA and TIC compounds in waterand air have been demonstrated.

The systems and methods discussed herein create a robust absorptiveenzyme support material from polymerizable pre-polymers to safely harborthe enzyme while maintaining its catalytic activity against CWAs andTICs. Enzymes are easily denatured and maintaining their catalyticactivity when supported on materials such as activated carbon hasheretofore been quite challenging. The systems and methods of variousembodiments of this invention may utilize pore forming pre-polymerswhich can be employed as a non-carbon based catalytic supports forenzymes selected to cause CWA and TIC degradation. In the event that theenzymes do get denatured, the enzyme can be reactivated even aftermultiple exposure cycles when supported on the innovative coatingsdiscussed above, e.g., such as polyurethane coating 42 includingβ-cyclodextrin (Poly-β-CD), trehalose (Poly-TH), and similar typematerials.

The systems and methods discussed above with reference to FIGS. 1-9 maymaximize loading and retention of catalytic enzymes by employingnon-conventional vaporous glutaraldehyde (GA) and its derivativesthereof in a controlled manner. This helps prevent enzymes from leachingout of the catalytic system during routine operations, which is a commonproblem typically experienced in conventional catalytic systems. One ormore embodiments of systems and methods of this invention include theability to sequentially incorporate the active components into amaterial, such as a fiber-based fabric, to create a comprehensive enzymepolymeric catalytic system using a unique deposition and chemicalmodification protocol. These polymeric enzyme systems (particles andfabrics) may provide the basis for full protection fabrics andprotective clothing and various articles for use against CWAs and TICssuch as chemical (both nerve and blister) agents and microbial attack.These system and methods discussed herein not only absorb CWAs and TICsupon encounter, but they preferably produce only non-toxic chemicals asa result of the decontamination.

The methods and materials of the systems and methods of the variousembodiments of this invention may be used to create a catalytic systemthat has enhanced activity, robustness, and reusability. The catalyticsystem may include a set number of coatings, or layers, built up on eachother, each providing protection from a different set of threat targets.In one example, the system starts out with the formation of an innerpolyurethane coating or layer consisting of β-cyclodextrin, trehalose,and calixarene. See, e.g., A. Singh, Yongwoo Lee, and Walter J.Dressick, Advanced Materials, 2004, vol. 16, pp 2112, incorporated byreference herein. This provides absorption towards CWA agents and TICsand acts as a stabilizing platform for catalytic enzymes. Thecyclodextrin in this layer acts as an absorbent capturing CWA and TICmolecules into its hydrophobic bucket-shaped molecule and the calixarenehas been shown to capture radiological waste particulates. Trehalose maybe included as a stabilizer for the enzymes. Trehalose most likelyprovides stability by slowing down enzyme denaturation under stressfulenvironments and as a moisture scavenger to deliver the necessary amountof water needed for the enzymatic decontamination reaction to occur.Once the polyurethane layer or coating is deposited, the biocatalystlayer may be placed onto the material, e.g., a fiber-based fabric. Thislayer preferably includes three decontaminating enzymes (e.g., OPH, OPAAand HD) preferably stabilized by monomeric trehalose and derivativesthereof. Once the enzymes are loaded, the entire system is exposed tovaporous GA for a period of time, which locks the enzymes into placewhile maintain their catalytic activity. Finally the system may becoated with chemically active polyethylenimine (PEI) and its derivativesthereof before being exposed to a second dose of vaporous GA to anchorthe PEI to the system and lock everything into place. These two vaporousGA treatments allow for the safe incorporation of the individual layersonto the fabric substrate and prevent them from leaching out or becominginactive during normal operations.

The systems and methods of the various embodiments of this invention areunique when compared to the currently available technologies. Twoprevious examples discussed in the Background section above forconstruction of enzymatic systems for the destruction of CWAs and TICsare LBL assembly and catalytic polymer nanosponge. The LBL systemdisclosed in U.S. Pat. No. 7,348,169, incorporated by reference herein,utilizes polyelectrolytes and natural polyionic interactions to build uplayers of catalytic enzymes and counter polyelectrolytes sequentially.This system uses the natural attractions of oppositely charged speciesto build up the layers. This allows the catalytic material to be builtup while retaining a relatively high percentage of catalytic activity inthe final product. The main drawback of the LBL approach is that becauseit uses ionic interactions, the system is easily delaminated losingmost, if not all, of its activity in a stressed environment. The NRLresearch team, inventors of U.S. Pat. No. 7,348,169, attempted toovercome this limitation by encasing the assembled catalyst structurewith a polymer net. Further, only a limited amount of enzymes can beloaded in the layer-by-layer assembly. The second system developed byUniversity of Pittsburgh utilizes organic solvents to polymerize theirmaterial in the presence of enzymes to create a nanosponge for catalyticdegradation. But because the enzymes are exposed to organic solvents avast majority of them become denatured and lose their activity. Thisgreatly limits the catalytic ability of the catalyst system.

As compared to the catalytic systems discussed above, the system andmethods of the various embodiments of this invention do not expose theenzymes to organic solvents and as such prevents their deactivation.Further, retention of the enzyme materials has been demonstrated throughcontrolled usage of vaporous GA as a binding agent to permanently attachthe enzymes and other active polymeric components onto the fabric,instead of using polyionic interactions. This results in greatlyextended working life of our catalytic system.

Example I Synzyme Coating

Amines are a well known group of weak nucleophilic compounds with a wideset of basic reactions which they participate in, but being relativelyweak nucleophiles they are not capable of reacting with organophosphatesand the like at ambient conditions, which requires a strongernucleophile for a reaction to occur. But even with such limitationstheir chemical properties and reactivities can be changed by taking themonomeric amines and polymerizing them into chains which contain a largeconcentration of primary, secondary and tertiary amines. These newpolymeric amines have been shown to exist in extensive branchednetworks. Branched polyethylenemine (BPEI), and shorter straight chainoligomeric amines, show an increased reactivity towards a wider spectrumof substrates. Once the amines are in this new polymerized form theyhave shown the ability to catalyze the hydrolysis of organophosphateswithout the amines themselves being used up in the reaction.

Laboratory experiments have shown that these amines, e.g., oligomericand branched polyamines, have a new capability to react withorganophosphates, such as Methyl Paraoxon (MPO) and Methyl Parathion(MPT), to generate p-Nitrophenol (pNP) which is less toxic then theoriginal analyte. The sample preparation was minimal and the reactivityof the final product was evident almost as soon as the target agent wasplaced onto the material. It was also shown that these amines were notbeing consumed during the hydrolysis reaction, but only acting as acatalyst. This may be referred to as a synthetic enzyme or “synzyme”.

A starting solution of the synzyme coating containing branchedpolyethyleneimine (BPEI) or a different oligomeric amine, andcyclodextrin bearing branched polyethyleneimine (CD-BPEI) was made bydissolving it in deionized (DI) water at a concentration of 10 mg/mL.After the BPEI/oligomeric amine, and cyclodextrin bearing branchedpolyethyleneimine (CD-BPEI) was completely dissolved, a fabric sample(cotton, Polyester/Polyamide (PE/PA)) was coated with the resultingsolution. The coating was done in 1 of 2 ways: (1) The sample wasforcefully impregnated by squeezing the synzyme solution into the fabricuntil it was saturated or (2) by spraying the synzyme solution with anatomizer onto the fabric sample until it was completely covered andallowing it to naturally impregnate the fabric. After the sample wascoated it was allowed to dry. The drying was also performed in one oftwo ways: (1) the sample was frozen and lyophilized or (2) the samplewas allowed to air dry by the use of air dryers. Both methods producedsamples showing the same rate of reaction.

Once the samples were ready they were tested against Methyl paraoxon(MPO) an organophosphate compound. In one example, testing was performedby placing a neat drop of the colorless MPO onto the synzyme coating 12of system 10, discussed above with reference to FIGS. 1-3, coated onfabric sample and allowing it to react with the coated material. Thetest results of this example are shown in FIG. 9. As shown, white cottonfabric 112 was coated with the synzyme solution consisting of BPEI andCD-BPEI followed by GA binding. After drying the fabric sample, a dropof MPO, which is clear, was placed onto fabric 112 at the area indicatedat 114. After exposure the sample developed a distinct yellow color,shown at 116. This is characteristic of the hydrolysis product,p-nitrophenol (pNP). The sample was then extracted using MeOH and theresulting solutions were analyzed using UV-VIS to conclusively prove thepresence of pNP by its distinctive peak at 405 nm, as compared to theMPO peak at 275 nm.

One issue which arises with the use of the BPEI is its inability to holdonto a chemical analyte long enough to allow the hydrolysis reaction tooccur, limiting its chemical activity. In order to increase thisactivity a system to increase the residence time of a target compoundnear the active site was developed. This was achieved through theutilization of a polycyclic compound (Cyclodextrin (CD)), as discussedabove with reference to FIG. 1, which possesses a hydrophobic pocketwhich can accept organophosphates through a guest-host complexformation. This combination of the reactive amine backbone and theretentive CD provides a dual attack strategy for not only reversiblycapturing organophosphates, and other similar compounds, but also forits hydrolysis into less toxic products.

While the reaction rate could be easily increased by the addition of theCD there was a second issue with using this as a reactive coating: Thereactive coating's dessolution in water. This issue was overcome bycross-linking the amine chains after deposition onto a substrate whichprevents its delamination. The simplest cross-linking agent used was adialdehyde compound, Glutaraldehyde (GA), which is highly reactive toamines. But it was also this reactivity which had to be controlled. If asolution of GA was to be exposed to a synzyme coated fabric, theresulting exhaustive cross-linking would make the final product verybrittle. This brittleness could be overcome by limiting the exposure ofthe GA to the fabric, which can be achieved by exposing the coatedfabric to only the fumes of GA (vaporized GA). The fumes provide enoughactive material to prevent the delamination of the BPEI coating but itprevents the extensive cross-linking which would lead to a brittleproduct. Another manner of controlling the amount of GA cross-linkingwould be to use a solution containing a mixture of monoaldehydecompounds along with GA. This prevents a complete cross-liking andprevents the material from becoming brittle.

The field of synthetic enzymes, or “enzyme mimics”, is currently underinvestigation. For example, the publication “An ‘Artificial Enzyme’Combining a Metal Catalytic Group and a Hydrophobic Binging Cavity” byRonald Breslow and Larry E. Overman, J. Am. Chem. Soc., 92 (4),1075-1077 (1970), incorporated by reference herein, discloses utilizingcycloexaamylose as a hydrophobic core provider to increase the residencetime of simple organic compounds to allow them to react with a metalbased catalyst. In another publication “High Rates and SubstrateSelectivities in Water by Polyvinylimidazoles as Transaminase EnzymeMimics with Hydrophobically Bound Pyridoxamine Derivatives as CoenzymeMimics,” by Rachid Skouta, Sujun Wei and Ronald Breslow, J. A. Chem.Soc. 131, 15604-15605, (2009), incorporated by reference herein,discloses using a polymer which contained polyaziridines to convertphenylpyruvic acid into phenylalanine in water, with an increase in thereaction rate of 3.5×10⁵ when compared to the reaction performed inwater with no polymer present.

Example II Sorption Reinforced Self-Decontaminating PolymericBio-Catalytic System Against Chemical Warfare Agents (CWA)

One purpose of one or more embodiments of this invention is to developsorption-reinforced, self-decontaminating enzyme-polymeric coatings formilitary uniforms (fabrics imbedded with particles) capable of providingcomprehensive protection at low and high temperatures against nerve,blister and microbial agents. Currently no known enzyme catalytic systemis available which allows for: 1) recycled use through self-repairing(refolding) technology, 2) to decontaminate chemical agents followed bysubsequent sequestering of break down products for safe disposal, 3)protection against blister agents (sulfur mustard) without producinggeno-toxic intermediates, and 4) increased protection by maximizing theloading and locking of catalytic enzymes and nucleophilic polymers atlow temperature environments (4° C.) as well as at ambient conditions.The system and method of one or more embodiments of this inventionprovides an efficiently decontaminating catalytic system which is highlycompatible with absorptive activated carbons and other decontaminatingtechnologies, and may be capable of providing comprehensive protectionagainst chemical nerve and blister agents.

In one example, biocatalyst components are embedded into the fabricwhich can protect against both nerve and blister agents. There is noknown efficient catalytic system that can decontaminate all classes ofchemical agents, while at the same time allowing for safe subsequentdisposal of the agents' reaction products, and that can be recycledthrough self-repair (i.e., enzyme refolding technology), and whichprotects against sulfur mustard or similar CWAs and TICs without theformation of genotoxic intermediates. Active enzymes were sequentiallyincorporated into particles and ultimately into fabric usingnon-covalent self-assembly to create a comprehensive catalytic system.It was demonstrated that the enzymatic system was capable of rapidresponse to and degradation of simulant challenges and was capable ofself-repair over multiple challenge cycles. The use of three fast actingdecontaminating enzymes (e.g., OPH, OPAA, and HD) incorporated onto apolymeric substrate (particles and fabrics) as the self-decontaminating,self-repairing, antimicrobial polymeric enzyme system highly catalyticnot only at ambient conditions but also at near-freezing temperatures.The enzyme-bearing polymeric substrates (particle and fabrics) typicallyconsist of four components. The catalytic enzymes are bound in anon-covalent fashion to the absorptive polymeric support viaphysisorption and then locked-in with the use of vaporous glutaraldehyde(GA) and thereby tuned favorably in the local microenvironment.Preferably, β-CD-BPEI is positioned at the outermost surface as acapping layer in the sorption reinforced self-decontaminating system.

Absorptive polymeric substrates have been prepared not only forabsorbing chemical agents by inclusion complex formation, but also foracting as insoluble supports to harbor enzymes safely. As a supportingmaterial of the OPH enzyme in the catalytic system of this invention,polyurethane-β-cyclodextrins (PU-β-CD) and polyurethane-trehalose(PU-TH) are used due to their sorption capabilities. These supportingmaterials are also flame-retardant, antimicrobial, catalytic in esterhydrolysis and capable of forcing chemical species into their cavitiesthrough hydrophobic interactions. In that manner, both PU-β-CD and PU-THcan sequester chemical agents.

Part I: Ultrathin, Compact Self-Decontaminating Catalytic Bio-PolymericSystem

The sorption reinforced catalytic system for the degradation of threatagents of one or more embodiments of this invention are preferablynon-toxic, non-corrosive, non-flammable, environmentally safe and highlycompatible with a number of different enzymes. Performance wasreproducible and the system and methods have been shown to be reusable,based upon testing against Soman (GD), Sarin (GB) anddiisopropylfluorophosphate (DFP) in a closed working environment and inopen air. In one example, the system and methods of one or moreembodiments of this invention discussed above, was stressed usingChlorox brand bleach, organic solvent (methanol) and 1M HCl solutionshowing a retention of 40%, 35% and 18% of original activity,respectively.

The sorption reinforced catalytic system and method for the degradationof threat agents of one or more embodiments of this invention isrecyclable and may be based upon the concept that decontamination occursonly in homogeneous local environments when chemical warfare agents(CWAs) and TICs get in contact with reactive species. CWAs and TICs aretypically hydrophobic. This may be overcome by the introduction ofhydrophobic bucket-shaped β-cyclodextrin, which helps extend theresidence time of the CWA molecule in the system through host-guestcomplex formation. Through this interaction a more favorable homogenousenvironment is formed allowing for the degradation of the hydrophobicchemical agent.

Activated carbon absorbs almost all pollutants indiscriminately but isnot recyclable. This leads ultimately to irreversible random attachmentwhich might block the reactive site of catalysts immobilized and thusprevents its easy access to supply anionic hydroxyl or amine groups,essential for hydrolytic degradation of pollutants.

The systems and methods of one or more embodiments of this invention arenot intended to create hypersorptive materials like activated carbons.Instead, absorptive recyclable polymeric matrixes are created whichfavorably harbor and stabilize enzymes by avoiding multi-pointattachment, and maximize the residence time of incoming CWAs and TICswithin the bucket-shaped cyclodextrin pores to allow enough time forthem to be degraded by the active layers of enzymes and nucleophilicβ-CD-polyethyleneimine (BPEI). The hydrophobic CWAs and TICs can bedegraded into smaller and less toxic hydrophilic by-products. Thoseabsorptive pre-polymeric materials can be easily created on fabrics atambient conditions simply by dipping the pre-polymerized organic solventmixture of β-cyclodextrin, hydroxypropyl β-cyclodextrin, calix[4]arene,calix[6]arene, calix[8]arenes, and spacers of hexyl diisocyanates (HDI)in DMF solvent.

Once CWAs and/or TICs come in contact with OPH-PU-β-CD, they getadsorbed to the ultrathin outermost surface where both catalytichydrolysis and nucleophilic substitution occur. Following thedegradation of the chemical agent, it's now hydrophilic byproducts arerepelled from the hydrophobic β-cyclodextrin interior and captured bytertiary (3°) amines present in the system of one or more embodiments ofthis invention, freeing the active site for another incoming CWAs andTICs molecules. This cycling allows for the continual capture,degradation and sequestering of by-products for safe disposal at aspecifically designated time by the user. FIG. 10 shows one example ofMPT toxin 120 degraded by enzyme coating 46 of system 40 discussed abovewith reference to FIGS. 4-6 to produce the degraded toxin pNP 122 whichis expelled from coating 46, as shown at 124.

Part II: Development of Absorptive and Nucleophilic PolyurethaneInvolving the Pore-Forming Building Blocks

Building blocks of the sorption reinforced catalytic system and methodfor the degradation of threat agents of one or more embodiments of thisinvention is now discussed. In one example, co-polymeric polyurethaneswere produced in-situ in the presence of pore-forming β-CD tostabilize/retain specific enzymes of interest by host-guest complexformation. Hydrolytic enzymes are employed to degrade CWAs and TICs.Branched polyethylene imines and oligomeric imines are used to degradeand sequester CWAs and TICs and their subsequent by-products:nucleophilic catalytic primary imines pointing towards the air ready toreact with incoming toxins, secondary imines used in cross linking forpermanent attachment to the fabric substrate, and tertiary iminesutilized as an active site to capture degraded by-products.β-cyclodextrins embedded throughout the system act as hosts to capturetoxins before eventually releasing then for decontamination. The entiresystem also shows antimicrobial activity, but actual active species isnot known as the individual components show no activity before coating.

II-1-1: Absorptive Polyurethane-β-Cyclodextrins (PU-β-CD): PreferentialAbsorption of Hydrophobic MPT Over Hydrophilic pNP

β-cyclodextrin and its derivatives are known to have exceptionalabsorption capabilities against TICs including CWAs. Los Alamos NationalLaboratory researchers developed a new class of organic nanoporouspolyurethane sponge (0.7-1.2 nm) using β-cyclodextrin as a buildingblock. These nanoporous polymers demonstrate excellent absorptioncapability against TICs in water. According to the report, hazardousorganic contaminants may be reduced to ppt levels in water by thesenanosponges.

In one example, polyurethane-β-cyclodextrin 130, FIG. 11, (PU-β-CD) wasprepared by exhaustive cross-linking of β-cyclodextrin 132 with a spacer134. According to published results and our experimental data,exhaustive cross-linking of cyclodextrin derivatives with excessiveamounts of alkyl spacers was not the method to generate the highlyabsorptive material with high surface area.

Using a carefully controlled and adequate amount of β-cyclodextrinimbedded in the cross-linked matrix by changing the amounts of reactivehexyl isocyanates (HDI) (equivalences of β-cyclodextrin vs. hexyldiisocyanate), the amount of enzyme loading of the enzyme layer couldalso be maximized accordingly. The uptake of both chemical species(p-nitrophenol and methyl parathion) by the enzyme layer 46 of system40, FIG. 4, and system 80, FIG. 7, was determined to demonstratepreferential sorption trends. In accordance with the systems and methodsof one or more embodiments of this invention, absorptive butnon-hyper-absorptive polymer containing Polyurethane-β-cyclodextrin(PU-β-CD)s were prepared as a physical support for enzymes, whileabsorptive towards toxins appropriate for safe disposal. PU-β-CDs arecapable of forcing chemical species into their hydrophobic cavities byhost-guest complex formation. As anticipated, experiments showedPolyurethane-β-CD coated with OPH enzyme showed a high sorption capacityfor methyl parathion (MPT) and its breakdown product p-nitrophenol(p-NP). Graph 140, FIG. 12, shows one example of p-NP and MPT absorptivebehavior of cotton thread coated with polyurethane layer 46, FIG. 4,including β-cyclodextrim as a function of hexyl diisocynate ratio.

II-1-2: Reactive Polyurethane-β-Cyclodextrin (R-PU-β-CD) asDecontaminating Synzyme

The PU-β-CD produced in accordance with one or more embodiments of thisinvention shows an unexpected ability to absorb and hydrolyze MPT within14 h, even in the absence of any enzyme catalyst.

In one example, samples from differently prepared Poly-β-CD were exposedto MPT for six days, the PU-β-CD material of one or more embodiments ofthis invention, and a control of stock MPT. Only the samples containingPoly-β-CD materials the material sorption reinforced catalytic systemfor the degradation of threat agents of this invention developed acharacteristic yellow color of p-nitrophenol (pNP) as a result ofdegradation, albeit over a much longer time frame (e.g., 6 days) thanthat in the presence of enzyme (e.g., a few minutes). The catalyticactivity became evident only after exposing β-CD in a cross-linkedpolymeric backbone, in absence of enzyme. In addition to their reactivecapability, when coated with OPH and then exposed to MPT vapor in thepresence of water vapor (100 μg), QNA cotton cloth functionalized withβ-CD turned yellow over 8 h at 40° C. (Not shown here), indicative ofthe qualitative breakdown of MPT.

II-2: Hygroscopic D-(+)-Trehalose and its Highly Absorptive PolymericDerivatives

D-(+)-trehalose is known to stabilize enzymes at low temperatures andwas also confirmed to stabilize enzymes during the immobilizationprocess. In one example, polyurethane-D-(+)-trehalose (PTH) was preparedin a specific molar ratio optimized for catalytic sequestration ofchemical warfare agents. This was prepared via coupling ofD-(+)-trehalose with HDI in DMF at 70° C. for 8 hours. The product wasthen freeze-dried over several days and the resulting powder was sieved.Characterization was carried out by FT-IR (the CO₂ group was observed at1725 cm⁻¹). The use of different amounts and types of alkyl spacers wereemployed to control the amount of trehalose incorporated in the polymermatrix, which subsequently affect the sorption capabilities andcatalytic ability when enzymes were incorporated, along with hydrolyticcapability against MPT.

These environmentally-benign, polymeric trehalose (PU-TH) molecules weredemonstrated to have high binding efficiency for the enzyme catalyst,while maintaining their chemical activity to efficiently hydrolyzeorganophosphates, and the ability to act as sorption-induced hydrolyzingvehicles against chemical threat agents.

II-3: Calix[8]Arene and its Polymeric Derivatives for Sequestration ofRadiological Waste

Polyurethane-calix[8]arene (PU-CX) was prepared, in a similarpreparation protocol of PU-β-CD, via coupling of calixarene with HDI inDMF at 70° C. for 14 hours. The OPH coated PU-CX particles were preparedand demonstrated to degrade MPT to pNP within about 5 minutes, as shownby graphs 162 and 164, FIG. 13. The particles of PU-CX could bepotentially usable for sequestration of radiological waste.

II-4: Synthesis of Co-Pre-Polymers: Co-PolymericPolyurethane-β-Cyclodextrin (PU-β-CD)/Poly-Urethane-D-(+)-Trehalose(PU-TH)/Polyurethane-Calix[8]Arene (PU-CX)

The enzyme based catalytic systems and methods of one or moreembodiments of this invention appear to be promising when coupled withabsorbing polymeric substrates (polyurethane-β-cyclodextrin (PU-β-CD),poly-calixarene (PU-CX) and polyurethane-D-trehalose (PTH)). In-situchemical modification and incorporating of highly absorbing butregenerative molecular unit such as co-polymeric polyurethane ofβ-cyclodextrin, calixarene, and polymeric D-(+)-trehalose were conceivedand demonstrated.

Environmentally-benign, light-weight polymeric pore-forming substrateswere prepared as a physical support to safely harbor enzymes.β-cyclodextrin and its derivatives are known to sequester CWAs and TICs,such as Soman (GD) and also to capture into their cavities biologicalspecies through inclusion complex formation with their hydrophobicpocket. Thus, incorporation of β-cyclodextrin into porous polymers mightimpart excellent sorption capacities and additionally could achieveclean “air” chemistry by (1) decontamination followed by (2)sequestration, when biological catalysts are incorporated into thepolymers. Its preferential sorption of MPT over pNP were also known.D-(+)-trehalose was demonstrated to protect enzymes and its hydrolyticand hygroscopic properties bring in moisture to the local environment.

Reaction of β-cyclodextrin, calixarene, and polymeric D-(+)-trehalose ina specific ratio with HDI produced insoluble absorptive particles viacoupling of β-cyclodextrin, D-(+)-trehalose and/or calixarene with HDIin DMF at 70° C. for 14 hours. These co-polymeric particles weredemonstrated to be chemically inert and robust enough to endure extendedexposure to highly acidic and highly alkaline media.

Part III Immobilization of DECON Enzymes to Absorptive PolymerSubstrates III-1. Stabilization of Enzymes with β-CD-BPEI and itsOligomeric Derivatives

The long term operational stability of any enzyme system is criticalbecause enzymes aggregate easily upon exposure to stressful conditions.Due to their amphoteric nature enzymes naturally degrade over time, butwithin the matrix the enzymes degrade more slowly even after cycled useover time under stressful conditions. In enzyme folding situations,chaperone molecules are known to assist them in reaching a specificconformation through different mechanisms.

OPH and a number of other enzyme systems have been shown to regain lostactivity under certain conditions. These include the use of a specificbuffered solution and the inclusion of “chaperone” molecules in enzymeformulations. The OPH-PU-β-CD cotton threads of the systems and methodsof one or more embodiments of this invention were demonstrated to havemuch greater activity after they were refolded in2-(N-Cyclohexylamino)ethane Sulfonic Acid (CHES) buffer solution andβ-cyclodextrin as a chaperone at low temperature (7° C.).

In one example, β-CD-BPEI cotton thread, e.g., about 1 m long, waswashed carefully with DMF followed by MeOH in a 50 mL size glass beaker.It was coated with OPH enzyme and used to degrade fresh MPT solution.The results were recorded, as shown by degrading kinetics curve 170,FIG. 14, (20 μM pNP after 30 min). After use, the OPH thread was washedusing CHES buffer briefly to eliminate MPT drops left on the thread. Afew drops of CHES buffer were added to the coated cotton thread, andthen placed in a freezer (7° C.). After 48 h, the thread was withdrawnfrom the freezer and exposed to a fresh 10 mL MPT solution to monitorthe subsequent catalytic degradation of “used” OPH thread. Curve 172shows the results of the catalytic activity as a result of the refoldingprocess.

This experimental result of enhanced activity using an artificialchaperone was also reproduced on OPH-nylon substrates (BiodyneB-membrane, Pall Corporation) when coated with OPH enzyme by knownprocedure. It was determined that once enzymes are anchored anartificial chaperone could serve to refold the enzymes incorporatedwithin the catalytic multilayer system. Both BPEI and CD-BPEI stabilizedOPH enzyme survived stressful conditions (e.g., rain, light, and heat),when coated by a certain protocol, meaning that BPEI and CD-BPEI serveas a chaperone to the OPH.

III-2. Stabilization of Enzymes with D-(+)-Trehalose and its OligomericDerivatives

D-(+)-trehalose and its derivatives were tested as a chaperone to repairdenatured OPH enzyme. D-(+)-trehalose, a dimeric glucopyranose, protectsand stabilizes the structure and activity of a variety of enzymes at lowtemperatures, and helps them to refold when denatured. Trehalose wasreported to display chaperone-like activity by stabilizing the nativeconformation of enzymes and protecting them from various kinds ofstresses. Along with β-CD and its derivatives, trehalose could stabilizethe enzyme within the matrix at ambient conditions and particularly atlow temperatures. The trehalose was employed during the drying of theOPH enzyme at low temperature and it also helped to stabolize it duringthe immobilization process, when cross-linked with vaporousglutaraldehyde and its derivatives.

III-3 Fixation of Enzymes by Polyimine Formation Using Vaporous GlutarDi-Aldehyde (GA) and its Derivatives

As was discussed above, the mode of enzymes immobilization is notlayer-by-layered assembly but a physical adsorption in presence ofenzyme-stabilizing D-(+)-trehalose, preferably followed by controlledvapor phase cross-linking via glutaraldehyde and its derivatives. Thespecific and selective cross-linking occurs preferably with secondaryamine groups to form imine bridges, as well as with some primary amines.In that manner, the “active” enzyme content could be increased in acontrolled manner. Layering of individual coated fabrics is anotherapproach for getting higher capacity for chemical absorption andcatalytic activity. Methods of attaching the particles and coatings havean impact on the reactivity of the system.

VI. End Capping Polymeric BPEI Having β-Cyclodextrins and Derivatives asMesogens. VI-1. β-CD-BPEI and its Polymeric and Oligomeric Derivativesof Ethylenimine (OEI)

Polyethyleneimine (PEI) is known to possess enzyme-like catalyticbehavior in organic reactions to breakdown phenyl esters at a highturnover rate. This may be employed as another method to bring oncatalysts in accordance with one or more embodiments of this invention.While the turnover rate for BPEI is lower than enzymes, BPEI'sresilience to stressful environments makes it a prime co-reactant inproviding comprehensive aerosol protection in all environments. Thisactivity was further increased through the formation of β-CD-BPEI whichallows for an even quicker reaction rate when compared to standard BPEI.FIG. 15 depicts the hypothetical catalytic processes of β-CD-BPEI forthe decontamination of the sarin (GB) toxin.

Branched PEI (BPEI) with alkyl groups, e.g., a propyl group or similartype group, are capable of forming a compact conformation to resistwater at the interfacial area and better served as nucleophilic chemicalmoieties than those of linear PEI (LPEI). One key element may be to makethe lone pair electrons of nitrogen group available in suchpolyethylenimine derivatives. Steric bulkiness of branched PEI may causeless substituted β-CD units, thus resulting in co-exiting ofnucleophilic primary (1°) amine, highly basic tertiary (3°) amine, whileallowing for an adequate amount of β-CD for hosting incoming toxins intoits interior. β-CD-BPEI may serve as a chaperone for OPH enzyme, apotential reactant for blood gases and toxic industrial materials, aneutralization reagent for acid gases, and may cause slow hydrolyticdegradation of chemical nerve agents upon encounter. For example, whentoxic sarin (GB) comes in contact with the system of one or moreembodiments of the invention, the GB will make a complex of inclusioninto β-cyclodextrin moiety, which maximizes residence time near reactivecomponent prior to attack of primary amine group at the fluorophosphonylgroup. The leaving fluoride anion thus could be removed by thenucleophilic amine group within the BPEI matrix. Also toxic“Hydrofluoric acid” (HF) breakdown product can be captured by highly“basic” tertiary amine group within the matrix for comprehensive andcomplete decontamination.

In one example, the β-CD-BPEI was prepared in aqueous medium at 70° C.from a reaction of branched polyethyleneimine (BPEI) withmonochlorotriazinyl-β-cyclodextrin (MCT-β-CD). In this example, theβ-CD-BPEI was prepared as electrospun nanofiber mats 174, FIG. 16,co-spun with poly(ethylene oxide) in a 1 to 1 ratio. Lyophilization ofthe mat allowed much of the open structure to be maintained, e.g., 200nm fiber diameter, as shown at 176.

In order to create these absorptive and reactive materials, branchedpolyethyleneimine (BPEI), e.g., having a molecular weight of about70,000, 20,000, 7,000, 600 Daltons, and the like, was treated in areaction vessel with monochlorotriazinyl β-cyclodextrin (MCT-β-CD) inalkaline aqueous solution (pH 8.0) at 90° C. for 18 h to produce β-CDbonded polyethylenimines derivatives. After the reaction, the resultingsolution turned a mild yellow to a yellowish-green color, depending onthe amount of β-CD bonded to the backbone of polymers. According to twoUV-VIS absorption spectra put in the same frame, the absorption band wasshifted from 230 nm to 374 nm. This indicates that attachment ofUV-active (but colorless by appearance) chlorotriazinyl β-CD to thebackbone of branched polyethylenimine progressed over time. The degreeof attachment and the processibility of the polymeric powders (yellowgreen) may be determined using standard protocol.

VI-2. Fixation of Reactive BPEI Using Vaporous Glutaraldehyde (GA) inGas Phase

Imine formation using glutaraldehyde (GA) is commonly employed inorganic and/or biochemistry. For example, GA-210, FIG. 17, preferablyreacts first with secondary amines then primary amines to bind BPEImolecules 211 and 213 together, as shown. The reacted GA-210 is shown at215. This has been demonstrated by mixing low level solutions of GA andBPEI together, and seeing the formation of particulates.

This complex formation is highly random and typically cannot becontrolled in water or organic solvent. However, it may be controlled bydiluting the amount of GA present in vapor phase for a brief exposure atambient conditions. To selectively attach enzymes into absorptivepolymers while avoiding multipoint attachment of enzymes, and to anchorβ-CD-BPEI subsequently to the enzymes layers in accordance with one ormore embodiments of this invention, a controlled attachment in gas phaseat ambient conditions was conceived and demonstrated. BPEI, as a baseand an acid scavenger, remains nucleophilic after GA treatment in gasphase at ambient conditions. Schiff Base formation between BPEI and GAis minimized in the vapor phase exposure as compared to reactions insolution.

The optimization of gaseous GA binding agent was performed to optimizeboth reactivity and retention of substrates to the fabric. For this asimple fume-chamber was utilized at room temperature in which liquid GAwas allowed to vaporize and generate fumes. A set of samples was placedinto the chamber and allowed to react for a set of pre-determined times.After the samples were taken out and dried their activity against MPTwas tested. FIG. 18 shows graphs 180 and 182 which represent the datacollected. The samples were left in the chamber from 30-720 minutes toget a good spread. Both sets of data, indicated by graphs 180 and 182,show that 2 hour (120 min) exposure of the fabric to GA fumes providesthe best activity while still maintaining flexibility and robustness ofthe fabric itself.

Through the utilization of gas phase coupling of secondary aminesbetween enzymes via glutaraldehyde (GA) to form inime bridges, theactive site is maintained allowing for the preservation of its chemicalactivity throughout the coating protocol, while at the same timeanchoring it to the substrate. At the same time, nucleophilic primaryamine groups remain intact as created in-situ in the capping layer ofCD-BPEI and its derivatives.

Testing of the System.

The sorption reinforced catalytic system for the degradation of threatagents of one or more embodiments of this invention may be based onembedding a sorption-rein-forced decontaminating layer of biocatalyticnanoparticles into the desired high-surface-area meltblown fabrics inorder to be able to decontaminate all classes of chemical agents whileat the same time allowing for safe disposal of the agents' reactionproducts via host-guest-complex formation. Active decontaminatingenzymes against G, V and H CWA agents may include organophosphoroushydrolase (OPH), organophosphorous acid anhydrolase (OPAA) andhaloalkane dehalogenase (HD). These enzymes were sequentiallyincorporated into highly absorbing polymeric substrates/particles andultimately onto fabrics to create a comprehensive catalytic system. Thecatalytic actions were performed through the use of highly absorbingpolymeric substrates to absorb CWA and TIC simulants and release theirbyproducts for safe disposal and subsequent recycled use. Theseenzyme-friendly self-decontaminating systems are an efficient platformfor destroying biological and chemical threat agents.

Status of Performance Testing

The sorption reinforced catalytic coating system and method for thedegradation of threat agents of one or more embodiments of thisinvention may be capable of decontaminating chemical nerve agents atambient conditions (temperature, moisture, and the like) in a closedenvironment, using a simplified flow-through air system in aqueousmedium and direct agent exposure without solvent medium, followed by thesequestering of breakdown byproducts for safe disposal at a later time.

The sorption reinforced catalytic coating system and method for thedegradation of threat agents may be incorporated onto cotton,cotton-nylon, modified nylon, meltblown and electrospun fiber-mats ofPE/PA, PU, and similar type materials, as discussed in the followingexamples from third party testing facilities.

In one example, degradation of nerve agent stimulant, e.g., more thanabout 10 g/m², was achieved using system 80, FIG. 7 with material 44configured as a meltblown (MB) fabric within a couple of hours ofexposure in aqueous medium. The treated MB fabrics 200, FIG. 19 ofsystem 80, FIG. 7, turned yellow, indicated at 202, FIG. 19, as a resultof decontamination. This indicates system 80 on the MB fabric absorbedthe breakdown product of the decontamination reaction (p-nitrophenol).This was reproduced using the same MB fabric during multiple runs. Thesame MB fabric also effectively degraded Soman (GD) as shown by graphs204, FIG. 20. In this example, measured concentration of the degradationproducts was fluoride.

Self-Decontaminating OPH-MB Nanofiber Mats

In one example, OPH enzyme was coated onto MB nanofibrous substratesemploying the coating system of one or more embodiments of thisinvention. The retention of the catalytic activity of the enzyme on thefibrous substrates were confirmed and reproduced by showing immediatedecontamination of the CWA simulant. See graphs 220 and 222, FIG. 21.

Testing by a third party, Assay Tech, Inc., Boardman, Ohio was performedon system 80, FIG. 7, deposited onto a fiber mat. These samples werechallenged with a constant concentration of CWA simulant MPO. The sampleshowed a capacity to sequester completely the challenge material for ashort time before a visible break though occurred. The recorded breakthough time is relatively short for these samples due to the highporosity of the fabric used. See graphs 260, FIG. 22.

Although specific features of the invention are shown in some drawingsand not in others, this is for convenience only as each feature may becombined with any or all of the other features in accordance with theinvention. The words “including”, “comprising”, “having”, and “with” asused herein are to be interpreted broadly and comprehensively and arenot limited to any physical interconnection. Moreover, any embodimentsdisclosed in the subject application are not to be taken as the onlypossible embodiments.

In addition, any amendment presented during the prosecution of thepatent application for this patent is not a disclaimer of any claimelement presented in the application as filed: those skilled in the artcannot reasonably be expected to draft a claim that would literallyencompass all possible equivalents, many equivalents will beunforeseeable at the time of the amendment and are beyond a fairinterpretation of what is to be surrendered (if anything), the rationaleunderlying the amendment may bear no more than a tangential relation tomany equivalents, and/or there are many other reasons the applicant cannot be expected to describe certain insubstantial substitutes for anyclaim element amended.

Other embodiments will occur to those skilled in the art and are withinthe following claims.

1. A reinforced catalytic coating system for the degradation of threatagents comprising: a polyurethane coating about a material configured toprovide loading and stabilization of one or more enzymes and for thesorption of the threat agents; an enzyme coating including the one ormore enzymes, the enzyme coating about the polyurethane coating andconfigured to degrade the threat agents; and a binding agent configuredfor enzyme immobilization to maximize loading and retention of the oneor more enzymes on the enzyme coating.
 2. The system of claim 1 in whichthe polyurethane coating is functionalized with organic bucket-shapedmolecules configured to stabilize the one or more enzymes of the enzymecoating.
 3. The system of claim 2 in which the bucket-shaped moleculesinclude cyclodextrin.
 4. The system of claim 3 in which the cyclodextrinand derivatives thereof include β-cylclodextrin.
 5. The system of claim4 in which the polyurethane coating is functionalized with chemicalgroups configured to stabilize the one or more enzymes of the enzymecoating.
 6. The system of claim 5 in which the chemical groups includesugar groups.
 7. The system of claim 6 in which the sugar groups includetrehalose.
 8. The system of claim 1 in which the polyurethane coating isfunctionalized with calixarene and derivates thereof configured for thesorption of radiological threat agents.
 9. The system of claim 1 inwhich the polyurethane coating is functionalized with chemical groups topromote water scavenging.
 10. The system of claim 9 in which thechemical groups include trehalose.
 11. The system of claim 1 in whichthe enzyme coating includes organophosphate degrading enzymes.
 12. Thesystem of claim 11 in which the organophosphate degrading enzymesinclude one or more of organophosphorous hydrolase (OPH),organophosphorous acid anhydrolase (OPAA) and haloalkane dehalogenase(HD).
 13. The system of claim 1 in which the vaporized binding agentincludes vaporous glutaraldehyde.
 14. The system of claim 13 in whichthe glutaraldehyde is vaporized.
 15. The system of claim 13 in which theglutaraldehyde is configured to selectively attach to the enzymecoating.
 16. The system of claim 15 in which the glutaraldehyde isconfigured to attach to the enzyme coating to prevent delamination. 17.The system of claim 1 in which the binding agent is configured toprovide for repeated cleaning cycle of the coating system.
 18. Thesystem of claim 1 in which the binding agent is configured to providefor reusability of the coating system.
 19. The system of claim 1 inwhich the enzyme coating is configured to degrade the threat agents inmoist environments.
 20. The system of claim 1 in which the materialincludes one or more of fiber based fabrics, meltblown nano basedfabrics, electrospun nano fibers, cotton, and/or nylon.
 21. The systemof claim 1 in which the coating system is configured to make protectiveclothing.
 22. The system of claim 1 in which the enzyme coating isexposed to a chaperone configured to enhance refolding of the one ormore enzymes.
 23. A method for making a reinforced catalytic coatingsystem for the degradation of threat agents, the method comprising:coating a material with a polyurethane coating configured to provideloading and stabilization of one or more enzymes and for the sorption ofthe threat agents; coating the polyurethane coating with an enzymecoating including the one or more enzymes configured to degrade thethreat agents; and exposing the material with the polyurethane coatingand the enzyme coating to a binding agent configured for enzymeimmobilization to maximize loading retention of the one or more enzymeson the enzyme coating.
 24. The method of claim 23 further including thestep of functionalizing the polyurethane coating with organicbucket-shaped molecules configured to stabilize the one or more enzymesof the enzyme coating.
 25. The method of claim 23 further including thestep of functionalizing the polyurethane coating with chemical groupsconfigured to stabilize the one or more enzymes of the enzyme coating.26. The method of claim 23 further including the step of functionalizingthe polyurethane coating with calixarene and derivates thereofconfigured for the sorption of radiological threat agents.
 27. Themethod of claim 23 further including the step of functionalizing thepolyurethane coating with chemical groups that promote water scavenging.28. The method of claim 23 further including the step of providing theenzyme coating with organophosphate degrading enzymes.
 29. The method ofclaim 23 further including the step of vaporizing the binding agent. 30.The method of claim 23 further including the step of making protectiveclothing with the coating system.